Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca(2+)-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles. Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.

Fibrosis in peripheral airways is responsible for airflow limitation in chronic obstructive pulmonary disease (COPD). Annexin A1 modulates several key biological events during inflammation. However, little is known about its role in airway fibrosis in COPD. We investigated whether levels of Annexin A1 were upregulated in patients with COPD, and whether it promoted airway fibrosis.

The incidence of asthma is increasing worldwide. Bronchial epithelium injury is common in asthma. The regulatory role of Annexin A1 (ANXA1) in bronchial epithelium injury is currently not well understood. The aim of the present study was to evaluate the role of ANXA1 on bronchial epithelium injury. The cell viability and levels of apoptosis were respectively tested by Cell Counting Kit‑8 and flow cytometry. Reactive oxygen species (ROS) content and the activity of oxidative indicators were assessed by commercial kits. Enzyme linked immunosorbent assay was performed to detect the activity of active caspase‑3. Reverse transcription‑quantitative polymerase chain reaction and western blot assays were used to determine the expression levels of the target factors. The results demonstrated that ANXA1 improved the viability of benzo[a]pyrene (Bap)‑treated bronchial epithelial cells. The Bap‑induced oxidative stress was mitigated by the reduction in ROS generation, and the regulation of the activity of superoxide dismutase, glutathione peroxidases, malondialdehyde and lactic dehydrogenase. In addition, apoptosis was decreased by ANXA1 via the reduction of the expression of B‑cell lymphoma 2 (Bcl‑2), and the increase in the expression of Bcl‑2‑associated X protein and cyclin D1. Furthermore, the expression of phosphatase and tensin homolog (PTEN) and focal adhesion kinase (FAK) was rescued and the phosphorylation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) was depressed by ANXA1, when compared with the Bap group. SF1670 treatment reversed the anti‑apoptotic effect of ANXA1. In conclusion, the results highlighted the protective effects of ANXA1 on bronchial epithelium injury, which most likely occurred via the PTEN/FAK/PI3K/Akt signaling pathway. Thus, the present study contributes to a potential therapeutic strategy for asthma patients.

Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18°C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen-glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-κB activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-κB activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-κB p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.

Chikungunya (CHIKV) is an arthritogenic alphavirus that causes a self-limiting disease usually accompanied by joint pain and/or polyarthralgia with disabling characteristics. Immune responses developed during the acute phase of CHIKV infection determine the rate of disease progression and resolution. Annexin A1 (AnxA1) is involved in both initiating inflammation and preventing over-response, being essential for a balanced end of inflammation. In this study, we investigated the role of the AnxA1-FPR2/ALX pathway during CHIKV infection. Genetic deletion of AnxA1 or its receptor enhanced inflammatory responses driven by CHIKV. These knockout mice showed increased neutrophil accumulation and augmented tissue damage at the site of infection compared with control mice. Conversely, treatment of wild-type animals with the AnxA1 mimetic peptide (Ac2-26) reduced neutrophil accumulation, decreased local concentration of inflammatory mediators and diminished mechanical hypernociception and paw edema induced by CHIKV-infection. Alterations in viral load were mild both in genetic deletion or with treatment. Combined, our data suggest that the AnxA1-FPR2/ALX pathway is a potential therapeutic strategy to control CHIKV-induced acute inflammation and polyarthralgia.

Annexin A1 (ANXA1) has been reported to promote tumor growth and resistance to chemotherapy drugs in lung cancer cells. In this study, we focused on the association of ANXA1 and chemosensitivity with a third generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), Osimertinib, in lung cancer cells with EGFR mutations. The overexpression of ANXA1 was observed in the lung cancer cells studied. The downregulation of ANXA1 with small interference RNA (siRNA) decreased the growth of lung cancer cells. In lung cancer cells with EGFR mutations, the knockdown of ANXA1 increased the chemosensitivity to Osimertinib, and decreased the tumorigenesis, invasion and migration of lung cancer cells. Further study showed that the knockdown of ANXA1 inhibited the phosphorylation of EGFR and down-stream Akt pathways and promoted apoptosis in lung cancer cells treated with Osimertinib. A mice xenograft lung cancer model was established in our study and showed that ANXA1 siRNA enhanced the effects of Osimertinib in vivo. Our study results showed that ANXA1 plays critical roles in chemosensitivity to EGFR-TKI in lung cancer cells with the EGFR mutation. Our efforts may be used in the development of lung cancer treatment strategies in the future.

Cisplatin is an antineoplastic agent widely used, and no effective treatments capable of preventing cisplatin-induced ototoxicity and neurotoxicity in humans have yet been identified. This study evaluated the effect of the anti-inflammatory annexin A1 (AnxA1)-derived peptide Ac2-26 in a cisplatin-induced ototoxicity model. Wistar rats received intraperitoneal injections of cisplatin (10 mg/kg/day) for 3 days to induce hearing loss, and Ac2-26 (1 mg/kg) was administered 15 min before cisplatin administration. Control animals received an equal volume of saline. Hearing thresholds were measured by distortion product otoacoustic emissions (DPOAE) before and after treatments. Pharmacological treatment with Ac2-26 protected against cisplatin-induced hearing loss, as evidenced by DPOAE results showing similar signal-noise ratios between the control and Ac2-26-treated groups. These otoprotective effects of Ac2-26 were associated with an increased number of ganglion neurons compared with the untreated cisplatin group. Additionally, Ac2-26 treatment produced reduced immunoreactivity on cleaved caspase 3 and phosphorylated ERK levels in the ganglion neurons, compared to the untreated group, supporting the neuroprotective effects of the Ac2-26. Our results suggest that Ac2-26 has a substantial otoprotective effect in this cisplatin-induced ototoxicity model mediated by neuroprotection and the regulation of the ERK pathway.

Annexin A1 (AnxA1) is an endogenous protein that modulates anti-inflammatory processes, and its therapeutic potential has been reported in a range of inflammatory diseases. The effect of AnxA1 on ischemia-reperfusion (IR)-induced lung injury has not been examined. In this study, isolated, perfused rat lungs were subjected to IR lung injury induced by ischemia for 40 min, followed by reperfusion for 60 min. The rat lungs were randomly treated with vehicle (phosphate-buffered saline), and Ac2-26 (an active N-terminal peptide of AnxA1) with or without an N-formyl peptide receptor (FPR) antagonist N-Boc-Phe-Leu-Phe-Leu-Phe (Boc2). An in vitro study of the effects of Ac2-26 on human alveolar epithelial cells subjected to hypoxia-reoxygenation was also investigated. Administration of Ac2-26 in IR lung injury produced a significant attenuation of lung edema, pro-inflammatory cytokine production recovered in bronchoalveolar lavage fluid, oxidative stress, apoptosis, neutrophil infiltration, and lung tissue injury. Ac2-26 also decreased AnxA1 protein expression, inhibited the activation of nuclear factor-κB and mitogen-activated protein kinase pathways in the injured lung tissue. Finally, treatment with Boc2 abolished the protective action of Ac2-26. The results indicated that Ac2-26 had a protective effect against acute lung injury induced by IR, which may be via the activation of the FPR.

Medication overuse headache (MOH) is a serious global condition. The interaction between headache attacks and medication overuse complicates the understanding of its pathophysiology. In this study, we developed a preclinical MOH model that incorporates these two key factors by overusing rizatriptan benzoate (RIZ, 4 mg/kg, i.g.) in a glyceryl trinitrate (GTN, 10 mg/kg, i.p.) induced chronic migraine mouse model. We observed that RIZ overuse aggravated GTN-induced cutaneous allodynia and caused a prolonged state of latent sensitization. We also detected a significant upregulation of Annexin-A1 (ANXA1), a protein mainly expressed in the microglia of the spinal trigeminal nucleus caudalis (SPVC), in GTN+RIZ mice. Intracerebroventricular injection of ANXA1-derived peptide Ac2-26 trifluoroacetic acid (TFA) (5 μg/mouse) inhibited bright light stress (BLS) induced acute allodynia via the formyl peptide receptor (FPR) in GTN+RIZ mice. These results suggest that ANXA1 may have an analgesic effect in triptan-associated MOH and could potentially serve as a therapeutic target.

Bladder cancer (BLCA) is one of the most common malignancies worldwide. One of the main reasons for the unsatisfactory management of BLCA is the complex molecular biological mechanism. Annexin A1 (ANXA1), a Ca2+-regulated phospholipid-binding protein, has been demonstrated to be implicated in the progression and prognosis of many cancers. However, the expression pattern, biological function and mechanism of ANXA1 in BLCA remain unclear.

Annexin A1 (ANXA1)-formyl peptide receptor (Fpr) system is potent effective mediators in the control of the inflammatory response. In this study, we evaluate the potential involvement of the Fpr family in the protective effect of the mimetic peptide of ANXA1 (ANXA12-26) using an experimental allergic conjunctivitis (AC) model in mice. Ovalbumin (OVA)/Alum-immunized wild-type (WT) and ANXA1-null (ANXA1-/-) Balb/c mice (days 0 and 7) were challenged by eye drops containing OVA on days 14-16, and two groups received ANXA12-26 alone or with Fpr antagonist Boc2 intraperitoneally during challenged days. As expected, plasma IgE anti-OVA levels increased significantly in the OVA-immunized WT and ANXA1-/- mice, supporting the efficacy of AC model. AC increased Fpr1 and Fpr2 levels in the conjunctiva and the lack of endogenous ANXA1 exacerbated Fpr2 expression only. In contrast, administering ANXA12-26 in the WT mice diminished Fpr2 levels in the conjunctiva, and the effect was reverted by Boc2. Ultrastructural analysis showed the co-localization of Fpr2 and ANXA1 in the plasma membrane of mast cells (MCs), eosinophils and neutrophils, supporting this system as being operative in the AC. Boc2 abrogated the ANXA12-26 effect by increasing the MC degranulation and the eosinophil influx in the conjunctiva, and these findings were supported by peroxidase eosinophil, eotaxin and MC protease levels. Additionally, the ANXA12-26-Fpr system in the AC was associated with the activation of ERK and JNK. Collectively, the data provided in vivo supports the anti-allergic effects of the ANXA1-Fpr system and may serve as a therapeutic target in this ocular disorder.

Uveitis is one of the main causes of blindness worldwide, and therapeutic alternatives are worthy of study. We investigated the effects of piperlongumine (PL) and/or annexin A1 (AnxA1) mimetic peptide Ac2-26 on endotoxin-induced uveitis (EIU). Rats were inoculated with lipopolysaccharide (LPS) and intraperitoneally treated with Ac2-26 (200 µg), PL (200 and 400 µg), or Ac2-26 + PL after 15 min. Then, 24 h after LPS inoculation, leukocytes in aqueous humor, mononuclear cells, AnxA1, formyl peptide receptor (fpr)1, fpr2, and cyclooxygenase (COX)-2 were evaluated in the ocular tissues, along with inflammatory mediators in the blood and macerated supernatant. Decreased leukocyte influx, levels of inflammatory mediators, and COX-2 expression confirmed the anti-inflammatory actions of the peptide and pointed to the protective effects of PL at higher dosage. However, when PL and Ac2-26 were administered in combination, the inflammatory potential was lost. AnxA1 expression was elevated among groups treated with PL or Ac2-26 + PL but reduced after treatment with Ac2-26. Fpr2 expression was increased only in untreated EIU and Ac2-26 groups. The interaction between Ac2-26 and PL negatively affected the anti-inflammatory action of Ac2-26 or PL. We emphasize that the anti-inflammatory effects of PL can be used as a therapeutic strategy to protect against uveitis.

Alzheimer's disease, characterized by brain deposits of amyloid-β plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-β levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-β and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-β load, due to increased clearance and degradation of amyloid-β by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

Extracellular vesicles (EVs) including plasma membrane-derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)-dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Host immune responses contribute to dengue's pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, interferon (alpha and beta) receptor one knockout (KO), AnxA1 KO, and formyl peptide receptor 2 (FPR2) KO mice were infected with dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac2-26 for analysis. In addition, the effect of Ac2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo. We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. Whilst the absence of AnxA1 or its receptor FPR2 aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestationsatteanuated the symptoms of the disease. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease.

Repair and regeneration of the injured skeletal myofiber involves fusion of intracellular vesicles with sarcolemma and fusion of the muscle progenitor cells respectively. In vitro experiments have identified involvement of Annexin A1 (Anx A1) in both these fusion processes. To determine if Anx A1 contributes to these processes during muscle repair in vivo, we have assessed muscle growth and repair in Anx A1-deficient mouse (AnxA1-/-). We found that the lack of Anx A1 does not affect the muscle size and repair of myofibers following focal sarcolemmal injury and lengthening contraction injury. However, the lack of Anx A1 delayed muscle regeneration after notexin-induced injury. This delay in muscle regeneration was not caused by a slowdown in proliferation and differentiation of satellite cells. Instead, lack of Anx A1 lowered the proportion of differentiating myoblasts that managed to fuse with the injured myofibers by days 5 and 7 after notexin injury as compared to the wild type (w.t.) mice. Despite this early slowdown in fusion of Anx A1-/- myoblasts, regeneration caught up at later times post injury. These results establish in vivo role of Anx A1 in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma.

Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse.

Preeclampsia (PE) is a disorder that is characterized by pregnancy‑induced hypertension. It has been reported that Annexin A1 (ANXA1) is highly expressed in the plasma of women diagnosed with PE. Therefore, the present study aimed to examine the effect of ANXA1 on PE rats. The PE animal model was constructed in rats using Nω‑nitro‑L‑arginine methyl ester (L‑NAME), and the blood pressure and urine protein levels of rats were detected. The pathological features of placental tissue, and the levels of inflammatory factors and ANXA1 were respectively measured by hematoxylin‑eosin staining, enzyme‑linked immunosorbent assay and immunohistochemical assay. The activity of trophoblasts obtained from PE placental tissue was measured using immunofluorescence staining, while cell apoptosis was assessed using flow cytometry. The levels of associated factors were determined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The results identified that systolic blood pressure, diastolic blood pressure, mean arterial pressure and urine protein levels were enhanced, and that the contents of ANXA1, tumor necrosis factor alpha (TNF‑α), interleukin (IL)‑1β, IL‑6 and IL‑8 were increased in the L‑NAME group. Transfection with small interfering RNA (siRNA)‑ANXA1 markedly decreased the apoptosis and inflammatory response of trophoblasts. In addition, siRNA‑ANXA1 upregulated the levels of B‑cell lymphoma‑2 (Bcl‑2) and pro‑caspase‑3, and downregulated the levels of Bcl‑2‑associated X protein, cleaved‑caspase‑3, TNF‑α, IL‑1β, IL‑6 and IL‑8. Furthermore, siRNA‑ANXA1 repressed the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3); however, siRNA‑ANXA1 did not alter the levels of JAK2 and STAT3. Therefore, silencing of ANXA1 suppressed the apoptosis and inflammatory response of PE rat trophoblasts, and downregulated JAK2/STAK3 pathway.

Annexin-A1 (AnxA1) and its N-terminal derived peptide Ac2-26 regulate the inflammatory response in several experimental models of disorders. This study evaluated the effect of endogenous AnxA1 and its N-terminal peptide Acetyl 2-26 (Ac2-26) on allergic asthma triggered by house dust mite (HDM) extract in mice. ANXA1-/- and wildtype (WT) mice were exposed to intranasal instillation of HDM every other day for 3 weeks, with analyses performed 24 h following the last exposure. Intranasal administration of peptide Ac2-26 was performed 1 h before HDM, beginning 1 week after the initial antigen application. ANXA1-/- mice stimulated with HDM showed marked exacerbations of airway hyperreactivity (AHR), eosinophil accumulation, subepithelial fibrosis, and mucus hypersecretion, all parameters correlating with overexpression of cytokines (IL-4, IL-13, TNF-α, and TGF-β) and chemokines (CCL11/eotaxin-1 and CCL2/MCP-1). Intranasal treatment with peptide Ac2-26 decreased eosinophil infiltration, peribronchiolar fibrosis, and mucus exacerbation caused by the allergen challenge. Ac2-26 also inhibited AHR and mediator production. Collectively, our findings show that the AnxA1-derived peptide Ac2-26 protects against several pathological changes associated with HDM allergic reaction, suggesting that this peptide or related AnxA1-mimetic Ac2-26 may represent promising therapeutic candidates for the treatment of allergic asthma.

The anti-inflammatory, pro-resolving annexin-A1 protein acts as an endogenous brake against exaggerated cardiac necrosis, inflammation, and fibrosis following myocardial infarction (MI) in vivo. Little is known, however, regarding the cardioprotective actions of the N-terminal-derived peptide of annexin A1, Ac2-26, particularly beyond its anti-necrotic actions in the first few hours after an ischemic insult. In this study, we tested the hypothesis that exogenous Ac2-26 limits cardiac injury in vitro and in vivo. Firstly, we demonstrated that Ac2-26 limits cardiomyocyte death both in vitro and in mice subjected to ischemia-reperfusion (I-R) injury in vivo (Ac2-26, 1 mg/kg, i.v. just prior to post-ischemic reperfusion). Further, Ac2-26 (1 mg/kg i.v.) reduced cardiac inflammation (after 48 h reperfusion), as well as both cardiac fibrosis and apoptosis (after 7-days reperfusion). Lastly, we investigated whether Ac2-26 preserved cardiac function after MI. Ac2-26 (1 mg/kg/day s.c., osmotic pump) delayed early cardiac dysfunction 1 week post MI, but elicited no further improvement 4 weeks after MI. Taken together, our data demonstrate the first evidence that Ac2-26 not only preserves cardiomyocyte survival in vitro, but also offers cardioprotection beyond the first few hours after an ischemic insult in vivo. Annexin-A1 mimetics thus represent a potential new therapy to improve cardiac outcomes after MI.